From the early part of this century, viruses have been used to treat cancer. The approach has been two-fold; first, to isolate or generate oncolytic viruses that selectively replicate in and kill neoplastic cells, while sparing normal cells. Investigators initially used wild type viruses, and this approach met with some, albeit limited success. While oncolysis and slowing of tumor growth occurred with little or no damage to normal tissue, there was no significant alteration in the course of the disease. See, Smith et al., Cancer 9: 1211–1218 (1956), Cassel, W. A. et al., Cancer 18: 863–868 (1965), Webb, H. E. et al., Lancet 1: 1206–1209 (1966). See, also, Kenney, S and Pagano, J. J. Natl. Cancer Inst., vol. 86, no. 16, p.1185 (1994).
More recently, and because of the reoccurrence of disease associated with the limited efficacy of the use of wild type viruses, investigators have resorted to using recombinant viruses that can be delivered at high doses, and that are replication competent in neoplastic but not normal cells. Such viruses are effective oncolytic agents in their own right, and further, can be engineered to carry and express a transgene that enhances the anti neoplastic activity of the virus. An example of this class of viruses is an adenovirus that is mutant in the E1 B region of the viral genome. See, U.S. Pat. No. 5,677,178, and Bischoff, J. R., D. H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna, L. Ng, J. A. Nye, A. Sampson-Johannes, A. Fattaey, and F. McCormick. 1996, Science. 274:373–6.
It is important to distinguish the use of replication competent viruses, with or without a transgene for treating cancer, from the second approach that investigators have used, which is a non-replicating virus that expresses a transgene. Here the virus is used merely as a vehicle that delivers a transgene which, directly or indirectly, is responsible for killing neoplastic cells. This approach has been, and continues to be the dominant approach of using viruses to treat cancer. It has, however, met with limited success, and it appears to be less efficacious than replicating viruses. Nevertheless, foreign genes have been inserted into the E1 region (see McGrory, Virology 163: 614–17 (1988)), the E3 region (see Hanke, Virology 177: 437–44 (1990) and Bett, J. Virol. 67: 5911–21 (1993)) or into the E3 region of an E1 deleted vector.
As mentioned above, to avoid damage to normal tissues resulting from the use of high dose viral therapy it is preferred that the virus have a mutation that facilitates its replication, and hence oncolytic activity in tumor cells, but renders it essentially harmless to normal cells. This approach takes advantage of the observation that many of the cell growth regulatory mechanisms that control normal cell growth are inactivated or lost in neoplastic cells, and that these same growth control mechanisms are inactivated by viruses to facilitate viral replication. Thus, the deletion or inactivation of a viral gene that inactivates a particular normal cell growth control mechanism will prevent the virus from replicating in normal cells, but such viruses will replicate in and kill neoplastic cells that lack the particular growth control mechanism.
For example, normal dividing cells transiently lack the growth control mechanism, retinoblastoma tumor suppressor, that is lacking in and associated with unrestricted growth in certain neoplastic cells. The loss of retinoblastoma tumor suppressor gene (RB) gene function has been associated with the etiology of various types of tumors. The product of this tumor suppressor gene, a 105 kilodalton polypeptide called pRB or p105, is a cell-cycle regulatory protein. The pRB polypeptide inhibits cell proliferation by arresting cells at the G1 phase of the cell cycle. The pRB protein is a major target of several DNA virus oncoproteins, including adenovirus E1a, SV40 large T Ag, and papillomavirus E7. These viral proteins bind and inactivate pRB, and the function of inactivating pRB is important in facilitating viral replication. The pRB protein interacts with the E2F transcription factor, which is involved in the expression of the adenovirus E2 gene and several cellular genes, and inhibits the activity of this transcription factor (Bagchi et al. (1991) Cell 65: 1063; Bandara et al. (1991) Nature 351: 494; Chellappan et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89: 4549.
The adenovirus, oncoproteins E1a, disrupts the pRB/E2F complex resulting in activation of E2F. However, neoplastic or normal dividing cells lacking sufficient functional pRB to complex E2F will not require the presence of a functional oncoprotein, such as E1a, to possess transcriptionally active E2F. Therefore, it is believed that replication deficient adenovirus species which lack the capacity to complex RB but substantially retain other essential replicative functions will exhibit a replication phenotype in cells which are deficient in RB function (e.g., normal dividing cells, or cells which are homozygous or heterozygous for substantially deleted RB alleles, cells which comprise RB alleles encoding mutant RB proteins which are essentially nonfunctional, cells which comprise mutations that result in a lack of function of an RB protein) but will not substantially exhibit a replicative phenotype in non-replicating, non-neoplastic cells. Such replication deficient adenovirus species are referred to as E1a-RB(−) replication deficient adenoviruses.
A cell population (such as a mixed cell culture or a human cancer patient) which comprises a subpopulation of neoplastic cells and dividing normal cells both lacking RB function, and a subpopulation of non-dividing, non-neoplastic cells which express essentially normal RB function can be contacted under infective conditions (i.e., conditions suitable for adenoviral infection of the cell population, typically physiological conditions) with a composition comprising an infectious dosage of a E1a-RB(−) replication deficient adenovirus. This results in an infection of the cell population with the E1a-RB(−) replication deficient adenovirus. The infection produces preferential expression of a replication phenotype in a significant fraction of the cells comprising the subpopulation of neoplastic and dividing normal cells lacking RB function (RB− cell) but does not produce a substantial expression of a replicative phenotype in the subpopulation of non-dividing neoplastic cells having essentially normal RB function. The expression of a replication phenotype in an infected RB(−)cell (neoplastic or dividing normal cells) results in the death of the cell, such as by cytopathic effect (CPE), cell lysis, apoptosis, and the like, resulting in a selective ablation of such RB(−) cells from the cell population. See, U.S. Pat. Nos. 5,801,029 and 5, 972, 706.
Typically, E1a-RB(−) replication deficient adenovirus constructs suitable for selective killing of RB(−) neoplastic cells comprise mutations (e.g., deletions, substitutions, frameshifts) which inactivate the ability of an E1a polypeptide to bind RB protein effectively. Such inactivating mutations typically occur in the E1a CR1 domain (amino acids 30–85 in Ad5: nucleotide positions 697–790) and/or the CR2 domain (amino acids 120–139 in Ad5, nucleotide positions 920–967), which are involved in binding the p105 RB protein and the p107 protein. Preferably, the CR3 domain (spanning amino acids 150–186) remains and is expressed as a truncated p289R polypeptide and is functional in transactivation of adenoviral early genes. FIG. 1 portrays schematically the domain structure of the E1a-289R polypeptide.
In addition to alterations in the E1a region of adenovirus, it would be desirable to enhance viral specific killing of neoplastic cells that lack RB function by constructing viruses that have critical replicative functions under the control of transcriptionally active E2F. The adenovirus replication cycle has two phases: an early phase, during which 4 transcription units E1. E2. E3, and E4 are expressed, and a late phase which occurs after the onset of viral DNA synthesis when late transcripts are expressed primarily from the major late promoter (MLP). The late messages encode most of the virus's structural proteins. The gene products of E1, E2 and E4 are responsible for transcriptional activation, cell transformation, viral DNA replication, as well as other viral functions, and are necessary for viral growth. See, Halbert, D. N., et al., 1985, J. Virol. 56:250–7.
If the adenoviral regions that are involved in virus replication could be brought under the control of E2F via an E2F responsive transcriptional unit, this would provide an enhanced adenovirus that selectively kills neoplastic cells that lack RB function, but not normal cells.
By way of background, the following references are presented relating to adenoviral vectors with alterations in regions involved in viral replication, including the E4 region, and E2F responsive promoters.
WO 98/091563, inventors Branton et al., presents methods and compositions for using adenoviral E4 proteins for inducing cell death.
Gao, G-P., et al., describe the use of adenoviral vectors with E1 and E4 deletions for liver-directed gene therapy. See. J. Virology, December 1996, p. 8934–8943.
WO 98/46779 describes certain adenoviral vectors capable of expressing a transgene comprising a modified E4 region but retaining E4orf3.
Yeh, P., et al describe the expression of a minimal E4 functional unit in 293 cells which permit efficient dual trans-complementation of adenoviral E1 and E4 regions. See, Yeh, P., et al J. Virology, January 1996, pages 559–565.
U.S. Pat. No. 5,885,833 describes nucleic acid constructs comprising an activator sequence, a promoter module, and a structural gene. The promoter module comprises a CHR region and a nucleic acid sequence that binds a protein of the E2F family.
Wang, Q. et al., in Gene Ther. 2:775–83 (1995) describe a 293 packaging cell line for propagation of recombinant adenovirus vectors that lack E1 and/or E4 regions. To avoid the transactivation effects of the E1A gene product in parental 293 cells as well as the over expression of the E4 genes, the E4 promoter was replaced by a cellular inducible hormone gene promoter, the mouse alpha inhibin promoter. Krougliak and Graham describe the development of cell lines that express adenovirus type 5 E1, E4, and pIX genes, and thus are able to complement replication of adenovirus mutants defective in each of these regions. See, Krougliak, V. and Graham, F., Human Gene Therapy, vol. 6: p. 1575–1586, 1995. Fang, B., et al. in J. Virol. 71:4798–803 (1997) describe an attenuated, replication incompetent, adenoviral vector that has the E4 promoter replaced with a synthetic GALA/VP16 promoter that facilitates packaging of the adenoviral vector in 293 cells that stably express the GAL4/VP16 transactivator. The virus was made replication incompetent by deletion of the E1 region of the virus.
U.S. Pat. No. 5,670,488 describes adenoviral vectors having one or more of the E4 open reading frames deleted, but retaining sufficient E4 sequences to promote virus replication in vitro, and having a DNA sequence of interest operably linked to expression control sequences and inserted into the adenoviral genome.
U.S. Pat. No. 5,882,877 describes adenoviral vectors having the E1, E2, E3 and E4 regions and late genes of the adenovirus genome deleted and additionally comprising a nucleic acid of interest operably linked to expression control sequences.
WO 98/13508 describes selectively targeting malignant cells using an E2F responsive promoter operably linked to a transgene of interest.
Neuman, E., et al., show that the transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter. See, Mol Cell Biol. 15:4660 (1995). Neuman, E., et al also show the structure and partial genomic sequence of the human E2F1 gene. See, Gene. 173:163–9 (1996).
Parr, M. J., et al., show that tumor-selective transgene expression in vivo is mediated by an E2F-responsive adenoviral vector. See, Nat Med. 3:1145–9 (1996). Adams, P. D., and W. G. Kaelin, Jr. show transcriptional control by E2F. See, Semin Cancer Biol. 6:99–108 (1995).
Hallenbeck, P., et al., describe vectors for tissue-specific replication. One such vector is adenovirus that is stated to selectively replicate in a target tissue to provide a therapeutic benefit from the vector per se, or from heterologous gene products expressed from the vector. In the former instance a tissue-specific transcriptional regulatory sequence is operably linked to a coding region of a gene that is essential for replication of the vector. Several coding regions are described including E1a, E1B, E2 and E4. See, WO 96/17053 and WO 96/17053.
Henderson, et al., in U.S. Pat. No. 5,698,443 shows an adenovirus vector having at least one of the genes E1A, E1B or E4 under the transcriptional control of a prostate cell specific response element.
It should be apparent that viruses offer another means for treating cancer. Thus, viruses that selectively replicate in, and kill neoplastic cells would be an invaluable weapon in a physician's arsenal in the battle against cancer.